东部马脑脊髓炎病毒荧光RT-PCR检测技术及标准质控品制备研究

Studies on the Development of Real-time Quantitative RT-PCR Assay for the Detection of Eastern Equine Encephalomyelitis virus And the Preparation of Reference Material

选取东部马脑脊髓炎病毒代表株进行序列比对分析,选择高度保守的区域,设计合成引物和Taq Man探针。对引物、探针浓度和反应条件进行优化,建立了东部马脑脊髓炎病毒荧光定量RT-PCR检测技术,实现了对东部马脑脊髓炎病毒的快速检测和鉴别。通过体外转录,制备了含有扩增区域核酸片段的东部马脑脊髓炎病毒核酸标准质控品cRNA,经稀释、分装定量、均匀性和稳定性检验后,作为方法的质控品对建立的方法进行评价,结果表明,应用建立的方法对阳性标准品的检测其灵敏度可达10eopies,对197份临床样品检测,表明建立的东部马脑脊髓炎荧光定量RT-PCR检测技术快速、敏感、特异。该方法的建立对该病的快速检测,及时采取相应防治措施、减少疫情散播有重要意义,也为动物及其产品的进出口检疫提供了一种可靠方法。

Nucleic acid sequences of Representative strains of Eastern Equine Encephalomyelitis virus （EEEV） were aligned with the DNAMAN software. Tile highly conserved internal region was then suhjected to the design of primers and probes. Real-time TaqMan RT-PCR assays were developed to detect and quantify EEEV by the selection and optimization of primers, plvbes and reaction conditions, Nucleic acid fragments covering amplification region were used to prepare cRNA as retrence materials. By in vitro transcription, the was detection limit of real-Lime RT-PCR was 10 copies per reaction using in vitro transcribed eRNA, By testing 197 clinical samples, It was demonstrated that the RT-PCR was a rapid, sensitive and reproducible diagnostic method. Therefore, the diagnostic methods will be useful for the prevention and reduction in the spreading of EEE. The study also provides a credible metilod for the rapid detection of EEEV from imported or exported animals and their products.